Drug resistant malaria kills 600,000 annually. Reversing horrific trends in incidence and mortality requires a balanced approach in vaccine and drug research, as well as field-based efforts to control vector populations and rates of infection. Current and future treatment of the many different strains of drug resistant Plasmodium falciparum malaria that now exist requires a more complete understanding of multiple genotypes and phenotypes. The struggle against drug resistant malaria is ongoing and must be met continuously, else we have learned nothing from the past 50 years while watching drugs fail. We must stay ahead of the resistance curve and define molecular mechanisms that guide ongoing drug and vaccine research. Our laboratory has helped to lead the field in the molecular level analysis of the PfCRT and PfMDR1 proteins, two transporters that are crucial for several forms of antimalarial drug and multidrug resistance. Recently, dozens of new isoforms of these proteins have been discovered, but only a handful have yet been studied. Elucidating function and resistance - conferring ability of these isoforms is crucial for defining local drug resistance and selecting region- and genotype-specific drugs whose efficacy is not compromised by local isoforms of these drug resistance transporters. In this grant period we will: Aim 1. Elucidate the molecular basis of PfCRT functional diversity and define K76T phenotypes. (1.1) Quantitatively compare all known PfCRT isoforms (1.2) Perform pfcrt gene editing experiments in P. falciparum to test in vitro conclusions, and Aim 2. Utilize a similar approach in order to elucidate PfMDR1 functional diversity, including: (2.1) Drug binding and drug transport in vitro, and (2.2) Pfmdr1 gene editing experiments in P. falciparum to test in vitr conclusions Aim 3. Elucidate the three-dimensional structures of PfCRT and PfMDR1 proteins. (3.1) Use a shot-gun crystallization strategy with multiple CRT orthologs, (3.2) Leverage tools within the TransportPDB resource, and (3.3) Generate tight-binding Nanobodies (Nbs) to chaperone crystal lattice formation.